J. Am. Chem. SOC 1993, 115, 4397-4398
1-Hydroxy-7-azabenzotriazole. An Efficient Peptide
Coupling Additive'
Louis A. Carpino'
Department of Chemistry
University of Massachusetts
Amherst, Massachusetts 01003
4397
the consideration that it incorporates within a single molecule
both key elements of the 1:l mixture of HOBt and a tertiary
amine which is of greater catalytic effect than HOBt itself in
couplings involving activeesters.I0 In fact, pronounced reactivity
enhancement was observed upon substitution of HOAt for HOBt
in a variety of circumstances, although 2, being insufficiently
basic to form stable salts in aqueous media, is not likely to adopt
the zwitterionic structure 3. If analogy to the reactions of HOBt
Received January 28, 1993
The most common peptide coupling additive is l-hydroxybenzotriazole (HOBt, l),I used either in combination with a
carbodiimide or another coupling agent or built into a standalone reagent such as 1-benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP)* or an analogous
uronium salt.3 Such additives generally inhibit side reactions
and reduce racemization. In this communication, l-hydroxy-
N'
I
I
OH
1
OH
2
7-azabenzotriazole (HOAt) (2) is described as a more efficient
additive which speeds up coupling processes, reduces the loss of
chiral integrity, and provides a visual indication4 (yellow-tocolorless) of the reaction endpoint.
While not new, HOAt6-*appears never to have been tested in
any coupling reaction. Initial examination of 2 was inspired by
' Abbreviations used: PG = protecting group; AA,, AA2 = amino acid or
dipeptide fragment; PS = Proton Sponge (l,S-bis(N,N-dimethylamino)naphthalene); EDC = N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide
hydrochloride; PCA =p-chloroaniline; DIEA = diisopropylethylamine; NMM
= N-methylmorpholine; DCC = dicyclohexylcarbodiimide;DCM = dichloromethane; DMF = dimethylformamide; Z = (benzy1oxy)carbonyl; Bz =
benzoyl; BOC = (tert-buty1oxy)carbonyl;Aib = a-aminoisobutyricacid; HOBt
= N-hydroxybenzotriazole; HOAt = 7-HOAt = 1-hydroxy-7-azabenzotriazole;
4-HOAt = 1-hydroxy-4-azabenzotriazole;HODhbt = 3-hydroxy-3,4-dihydro4-oxo- 1.2.3-benzotriazine; BOP = 1-benzotriazolyloxytris(dimethy1amino)phosphonium hexafluorophosphate; HBTU = 0-(benzotriazol- l-y1)-1,1,3,3tetramethyluronium hexafluorophosphate; HATU = O-(7-azabenzotriazoll-yl)-l,l,3,3-tetramethyluroniumhexafluorophosphate; TATU = 047azabenzotriazol- 1-yl)- l11,3,3-tetramethyluronium
tetrafluoroborate; HBPyU
= 0-(1 H-benzotriazol-l-yl)-l,l,3,3-bis(tetramethylene)uroniumhexafluorophosphate; HAPyU = O-(7-azabenzotriazol-l-yl)l11,3,3-bis(tetramethy1ene)uronium hexafluorophosphate.
(1) (a) Konig, W.; Geiger, R. Ber. Dtsch. Chem. Ges. 1970, 103,788. (b)
Kdnig, W.; Geiger, R. Ber. Dtsch. Chem. Ges. 1970, 103, 2024, 2034.
(27 Review: -Le Nguyen, D.; Castro, B. Peptide Chemistry 1987, Proc.
Jpn. Symp. Pept. Chem. 1988, 231.
(3) Knorr, R.; Trzeciak, A.; Bannworth, W.; Gillessen, D. Tetrahedron
Lett. 1989, 1927.
(4) Theadditive HODhbt also yieldsa yellow-coloredanion.5 Moreeffective
in reducing racemization than HOBt, this additive has had limited use due
to the possible Occurrence of side reactions involving attack at the carbonyl
function. Comparisons with HOAt show that HODhbt is less effective in
both reactivity enhancement and inhibition of racemization. According to
Azev and co-workers,",' the pK., values of HOBt, 4-HOAt, and 7-HOAt are
4.60, 3.02, and 3.28, respectively. These figures could account for some but
not all of the special properties of HOAt and its derivatives.
( 5 ) Atherton, E.; Cameron, L.; Meldal, M.; Sheppard, R. C. J. Chem.
SOC.,Chem. Commun. 1986, 1736.
(6) (a) Azev, Y.; Mokrushina, G. A.; Postovoskii, I. Ya.; Sheinker, Yu. N.;
Anisimova, 0. S. Chem. Heterocycl. Compds. 1976, 1172. (b) Mokrushina,
G. A,; Azev, Y.; Postovoskii, I. Ya. Chem. Heterocycl. Compds. 1975, 880.
(c) Azev, Y.; Mokrushina, G. A,; Postovoskii, I, Ya. Chem. Heterocycl.
Compds. 1974, 687. (d) Sacher, R. M.; Alt, G. H.; Darlington, W. A. J .
Agric. Food Chem. 1973, 21, 132.
(7) Previously obtained from 3-fluoro-2-nitropyridine by reaction with
hydrazine, the synthesis of 2 has been simplified by methylation [Yutilov, Y.
M.; Ignatenko, A. G. Khim. Prom-st., Ser. Reakt. Osobo Chist. Veshchestva
1981.27 (Chem. Abstr. 1982,96,68773s)] of 2-nitro-3-pyridinol followed by
treatment of the methyl ether with excess hydrazine according to the method
described by Azev et al." for the corresponding fluoride. Both HOAt and
HATU are available from Millipore Corporation, Bedford, MA 01 730.
0002-7863/93/ 1515-4397$04.00/0
3
k
4
is taken as a guide, increased efficiency of 2 relative to 1 as a
coupling additive could be governed by the formation or reactivity
of an active ester intermediate. Although precise mechanistic
details need yet to be established, reactions of HOAt esters appear
to be enhanced relative to those of the HOBt analogs, and it is
tempting to postulate the importance of the neighboring group
effect depicted in 4 as an important factor.ll3l2 As an example,
the highly hindered secondary amine SI4undergoes acylation by
2-phenylpropanoic acid within about 22 h in the presence of HOAt
under conditions which with HOBt provide only a trace of the
mixed diastereomeric amides. With the cis-isomer of 5, acety-
csr 5
5
csr 5
6
lation via acetic acid in the presence of HOAt and EDC gives
within 30-50 min the corresponding amide, mp 162-164 OC, in
87% yield, whereas the analogous reaction with HOBt is still
(8) Assignment of structure (C/N/O skeleton) to HOAt and its 4-isomer
follows from the synthetic methods adopted and analogy to HOBt syntheses.
The active ester intermediates generated during acylation reactions involving
these additives may participate in the same kinds of equilibria previously
observed for the analogous esters derived from HOBL9
(9) Cf.: Barlos, K.; Papaioannou, D.; Voliotis, S.; Prewo, R.; Bieri, J. H.
J. Org. Chem. 1985, 50, 696 and references cited therein.
(10) See: Carpino, L. A.; Chao, H. G.; Beyermann, M.; Bienert, M. J.
Org. Chem. 1991, 56, 2635 and references cited therein.
(1 1) Similar interactions have been proposed to account for the reactivity
of esters of 8-hydroxyquinoline relative to that of the 3- and 6-isomers.'2 ' 3
There is no evidence to date that the isomeric 1-hydroxy-4-azabenzotriazoleh"
shares all of the unique properties of the 7-aza derivative 2. Thus, for the
4-aza compound color monitoring is possible and coupling rates are enhanced
although racemization is not substantially reduced relative to the case of
HOBt. In the reaction of the highly hindered a,a-disubstituted amino acid
Z-Aib-OH with the weakly-basic aromatic amine p-chloroaniline (PCA) in
DMF-d,, rough NMR measurements show that about half of the PCA
disappears into the corresponding amide within 4 N . 5 min for 7-HOAt and
65-70 min for 4-HOAt, although reaching this stage requires over 24 h in the
case of HOBt. About 260 min are required for comparable reactivity in the
presence of the well-known additive HODhbt (see also footnote 4).
(12) (a) Jakubke,H.-D.;Voigt,A. Chem. Ber. 1966,99,2419. (b) Jakubke,
H.-D.; Voigt, A.; Burkhardt, S. Chem. Ber. 1967, 100, 2367.
(13).For a deliberate although unsuccessful attempt to design an HOBt
derivative for which intramolecular base catalysis by a neighboring sulfonate
function might enhance reactivity and suppress racemization during peptide
coupling, see: Savrda, J.; Ziane, N.; Guilhem, J.; Wakselman, M. J . Chem.
Res. ( S ) 1991, 36.
(14) Carpino, L. A. J. Chem. Soc., Chem. Commun. 1966, 858.
1993 American Chemical Society
4398 J. Am. Chem. Sot., Vol. 115, No. 10, 1993
Communications to the Editor
Table 1. Loss of Chirality During Coupling of PG-AAI-OH with H-AA2-OMeHCl
HOAt"
or H-AA2-OMe in D M F in the Presence of HOBt and
run
PG
AA I
AA2
conditions
oL-isomer (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Z
Z
Z
Z
Z
Z
2
Z
Z
Phg
Phg
D-Phg
Phg
Phg
Phe-Val
Phe-Val
Phe-Val
Phe-Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Val
Ala
Ala
Ala
Ala
Val
Val
Val
Val
Val
Val
Val
Val
HOAt, PS (1 equiv), EDC, 9 hb
HOBt, PS (1 equiv), EDC, 9 hb
HOAt, PS (1 equiv), EDC, 6 h
HATU, PS (2 equivs), 7 h
HBTU, PS (2 equivs), 7 h
HOAt, N M M (1 equiv), EDC, 1.25 h
HOBt, N M M (1 equiv), EDC, 2.25 h
HATU, DIEA (2 equivs), 3.5 h
HBTU, DIEA (2 equivs), 4 h
HOAt, N M M (1 equiv), EDC, 20 h
HOBt, N M M (1 equiv), EDC, 20 h
DCC, 24 hc
HOAt, DCC, 24 hC
HOBt, DCC, 24 hc
HOAt, DCC, DCM solvent, 24 hc
HATU, N M M (2 equivs), 3 h
HBTU, N M M (2 equivs), 3.5 h
<I-2
3.7
<1-2
< 1-2
4.0
<I-2
4.1
< 1-2
4.1
28.1
45.4
61.5d
14.4
41.9
<1-2
28.3
46.8
BZ
BZ
BZ
BZ
BZ
BZ
BZ
Bz
Test couplings were carried out by preparing a solution of 0.37 mmol of a protected amino or dipeptide acid, 0.33 mmol of HOAt or HOBt, and
0.33 mmol of an amino acid ester (or its hydrochloride plus an equivalent amount of a tertiary amine) in 1 mL of DMF. The mixture was cooled in
an ice bath and treated with 0.37 mmol of DCC or EDC. For reactions involving uronium salts, 0.74 mmol of a tertiary amine was substituted for
the HOAt or HOBt. For HOAt reactions in the absence of excess amino component or tertiary base, disappearance of the yellow color signaled
completion of the coupling process, and workup proceeded soon thereafter. All reactions were carried out in DMF, except in the case of run 15 where
DCM was used. Generally stirring was continued in the ice bath for 1.5-2 h and then a t room temperature for the times indicated. Dilution with
15 mL of CH2C12 and 200 mL of water was followed by extraction with 4-5 IO-mL portions of CHzCl2 and washing in order with 10"
portions
of 10% HCI (twice), H 2 0 (once), and 0.5 M N a H C 0 3 (twice). Drying and removal of solvent gave the crude peptide (generally in yields of 70-90%),
which was examined by 200- or 300-MHz IH N M R for the presence of diastereomeric contamination (OMe and/or MeCH). In a few cases, the IH
N M R analyses were confirmed by H P L C analysis. Thus for runs 6 and 7 H P L C analysis showed 0.3% and 4.7% of the LoL-isomer, respectively.
Conditions for the separation of this set of diastereomers have been described by Miyazawa and co-workers.19 Under the same conditions, 4-HOAt
and HODhbt led to 6.1% and 4.1% of the oL-form, respectively. In these cases (runs 12-15), H-Val-OMeHCI was converted to the free base by
treatment with concentrated K2CO3 and extraction into CH2Cl2 prior to addition to the reaction mixturd. This figure is close to that reported as arising
from the racemic oxazolone derived from N-benzoylvaline (65% DL), thus demonstrating the importance of asymmetric induction in the case of this
particular coupling.2°
*
incomplete after 7.5 h. The coupling of adjacent Aib residues
is known to be difficult.15 When BOC-Aib-OH is coupled to
H-Aib-OMe in the presence of HOBt, reaction is incomplete
after 24 h (ca.35%oftheHOBtesterremainsunreacted),whereas
with HOAt a theoretical yield of the dipeptide is obtained.
Similar enhanced reactivity is shown by uronium salt 7
(HATU)16-'7relative to its HOBt analog HBTU.3 Table I collects
NMe2
7
examples which illustrate the effectiveness of 2 in reducing
racemization. One model system which is easy to follow via IH
N M R techniques involved coupling of a urethane-protected
derivative of the sensitive nonproteinogenic amino acid a-phenylglycine.'g Upon treatment of the (benzy1oxy)carbonyl de(15) Coupling reactions involving protected Aib which are carried out in
the presence of HOBt often lead to the isolation of stable OBt esters. See,
for example: Fr6rot. E.; Coste, J.; Pantaloni, A.; Dufour, M.-N.; Jouin, P.
Tetrahedron 1991, 47, 259.
( 1 6) In honor of the originators of these reagents, suggested abbreviations
for these new modifications will keep to the earlier style in spite of the fact
that the word order in the French language reverses that of the English
equivalents. Thus, HATU for 7, HAPyU for its pyrrolidine analog, TATU
for the tetrafluoroborate analog of 7, etc. The IH-NMR spectra of these
compounds exhibit the highly characteristic ABX/AMX pattern (threedouble
doublets) of a pyridine nucleus disubstituted by electron-withdrawing groups
at the 2,3-positions. Thus HOAt (CDC13-DMSO-d6): 8 7.35 (dd, 1, 8-H),
8.3 (dd, I , y-H), 8.66 (dd, 1, wH); J , , , ,= 4.2 Hz, J,j,y 8.2 Hz, .Ic,,?
= 1.6
Hz. HATU (DMSO-dc,): 6 3.2 (d, 12, CH,), 8.0 (dd, 1, 8-H), 8.45 (dd, 1,
y-H), 8.9 (dd, 1, wH), J,,p = 4.4 Hz,
= 8.4 Hz, = 1.8 Hz.
( 17) Prepared from tetramethylchloroformamidiniumhexafluorophosphate
by analogy to a published method [Dourtoglou, V.; Gross, B.; Lambropoulou,
V.; Zioudrou, C. Synthesis 1984, 5721.
rivative with valine methyl ester hydrochloride in the presence
of HOBt and 1 equiv of PS or with HBTU and 2 equivs of PS,
3 . 7 4 0 % of the DL-diastereomer was formed (runs 2 and 5). This
was reduced to less than 1-2% by substitution of HOAt for HOBt
in these reactions (runs 1 and 4).
A second example, relevant to the technique of segment
coupling, involved reaction of Z-Phe-Val-OH with alanine methyl
ester.I9 With this system, HOBt or HBTU coupling (runs 7 and
9) in the presence of N M M or DIEA gave 4.1% of the LDLisomer. Again in this case the use of HOAt or HATU lowers the
extent of racemization to less than 1-2% (runs 6 and 8). Finally,
the highly sensitive coupling of benzoylvaline with valine methyl
ester20 shows that HOAt reduces racemization to about onethird or one-half the level found for comparable HOBt reactions
(runs 10-17). For thissystem,evenwithHOAt, onlyinanonpolar
solvent such as dichloromethane was it possible to effect coupling
without detectable racemization.
Additional applications of HOAt and reagents derived therefrom to both solution and solid-phase coupling reactions, ester
formation, etc., as well as modification of the basic structural
elements deemed responsible for the unique nature of these systems
(neighboring group effects), are under active scrutiny.
Acknowledgment. We are indebted to the National Institutes
of Health (GM-09706) and the National Science Foundation
(CHE-9003192) for support of this work, which was carried out
during the tenure of a University of Massachusetts Faculty
Fellowship. Drs. Chuck Phoebe, Steven A. Kates, Michael
Schiimann, and Dieter Runald are thanked for some of the N M R
and HPLC data.
(18) Carpino, L. A. J . Org. Chem. 1988, 53, 875.
(19) Yamada, T.; Kurokawa, S.; Dejima, K.; Watanabe, K.; Kotani, M.;
Miyazawa, T.; Kuwata, S. Mem. Konan Uniu., Sci. Ser. 1985, 32, 11.
(20) Miyazawa, T.; Otomatsu, T.; Yamada,T.; Kuwata, S. Chem. Express
1991, 6, 61.